We are analyzing the interactions between virus glycoproteins and their receptors for several enveloped viruses important for human health. Our goals are to define mechanisms of virus fusion and entry, elucidate mechanisms of virus tropism, identify cellular receptors, develop strategies to elicit and detect neutralizing antibodies, and develop novel therapeutic and preventative approaches. 1) Human herpesvirus 8 (HHV-8) is associated with Kaposis sarcoma, a major opportunistic disease associated with HIV infection. We established a specific fusion assay between cells chronically infected with HHV-8 and various target cells. During the past year, we established an HHV-8 fusion assay using recombinant glycoproteins; gB, gH and gL were found to be essential; K8.1, while not required, augmented the fusion signal. Anti-peptide sera reactive with each of these glycoproteins have been obtained. In our effort to develop a system to isolate the HHV-8 receptor, we have obtained a cDNA library (in vaccinia) from a highly fusion-permissive cell line; moreover we have developed a reporter gene containing several useful elements for selection/screening of fused cells: extracellular HA and myc tags (for selection by magnetic beads) and intracellular GFP (for screening, and possilby for selection by FACS). Optimal conditions for a fusion-dependent library screen are under development. 2) Flaviviruses. We are particularly interested in developing systems to study HCV entry, for use in assaying neutralizing antibodies as well as for isolation of the HCV receptor. To this end, we seek to produce flavivirus subviral particles particles (SVPs) containing the E1 and E2 glycoproteins on their surface. One approach is to exploit a recombinant baculovirus system, which to date is the only approach that has yielded large amounts of purified HCV SVPs. We seek to encapsidate an RNA encoding a reporter gene that would be activated upon entry of the particles into target cells. Because of the unavailability of a cell system that supports HCV replication, we are also analyzing West Nile virus which is much more experimentally acceptable. Methods developed for WNV will be applied to HCV.. 3) SARS coronavirus. We are participating in a collaborative effort to study SARS coronavirus entry, using the recombinant S glycoprotein. A recombinant vaccinia virus has been produced and shown to express surface S. Efforts are underway to develop entry/fusion assays, using either cell fusion or pseudotyped retroviral particles.